student research day - Case Western Reserve University School of ...

Michael J. Tchou
Mutations in GJA5 may lead to atrial fibrillation
Michael J. Tchou, Rob Wirka, Mina Chung, Dave Van Wagoner, Stela Berisha, Priya Malik, Brandon DuGar and Jonathan
Smith Ph. D.
Cell Biology Department
Cleveland Clinic Lerner Research Institute
Introduction: Atrial fibrillation (AF) affects 1 in every 25 people over the age of 60 in the United States. While a large
proportion of AF is caused by structural changes, 30% of cases lack a structural cause (lone AF) and may have a genetic
component to their etiology. Recent research has suggested that mutations in GJA5, the gene encoding the gap junction
protein Connexin 40 may be implicated in the development of AF. A case-control comparison of single nucleotide
polymorphisms of the GJA5 gene may reveal significant mutations.
Methods:
Blood derived or atrial tissue derived DNA was prepared from a cohort of 200 lone AF subjects. For the detection of rare
variants in the GJA5 gene, the three exons and flanking sequences were amplified and sequenced. For the detection of
common single nucleotide polymorphisms (SNPs) in the GJA5 gene, the DNA was subjected to an Illumina SNP
microarray and/or assay of single SNPs by the TaqMan procedure. GJA5 transcript levels were determined from atrial RNA
using an Illumina gene expression microarray.
Results:
Two novel heterozygous mutations were identified among 24 lone AF samples through DNA sequence analysis. In
addition a common 25 bp insertion-deletion variation in the 3‖ UTR that was discovered in a related project was verified
in these tissues and was associated with the level of the GJA5 transcript in atrial tissue. Experience was gained in
laboratory procedures including DNA extraction and preparation, gel electrophoresis, PCR, primer design, sequencing,
sequence analysis, and microarray analysis.
Conclusions:
Early results indicate that variations in the GJA5 gene appear to be related to GJA5 expression levels and incidence of
AF. Ongoing sample analysis will allow a more definitive association of GJA5 genetic variation and AF.
Supported by T-35
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